Up to now, a large number of microorganisms have been known to store polyester as an energy source substance within cells. A typical example of the polyester is poly-3-hydroxybutyric acid (hereinafter referred to briefly as “P(3HB)”). P(3HB) is a thermoplastic polymer and is biodegradable in the natural environment and, thus, has recently attracted attention as an ecofriendly green plastic. However, since P(3HB) is high in crystallinity, it is hard and fragile, so that the range of practical application thereof is limited. Therefore, studies have been undertaken to modify the P(3HB) for improving these properties.
In the course of the study, a copolymer P(3HB-co-3HV) derived from 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (hereinafter referred to briefly as “3HV”), and a production method thereof have been developed (Japanese Kokai Publication Sho-57-150393, Japanese Kokai Publication Sho-59-220192 and Japanese Kohyo Publication Hei-11-500008). This P(3HB-co-3HV) is rich in flexibility as compared with P(3HB), hence it was considered to have a wide application range.
Methods for producing the copolymer P(3HB-co-3HV) described in these patent documents comprise growing cells in the first stage and culturing a microorganism with restricting nitrogen or phosphorus in the latter stage to produce the copolymer similarly to the conventional methods for producing P(3HB).
Moreover, as for P(3HB-co-3HV), since the flexibility changes as a content of 3HV increases, researches for controlling the 3HV content have also been made. For example, propionic acid is used in Japanese Kokai Publication Sho-57-150393 and Japanese Kokai Publication Sho-63-269989, and propan-1-ol is used in Japanese Kokoku Publication Hei-7-79705, and by changing an addition amount thereof to a medium, the 3HV content in P(3HB-co-3HV) is controlled to produce P(3HB-co-3HV) having the 3HV content of 10 to 90 mol %.
Actually, however, P(3HB-co-3HV) shows only slight changes in the characteristics even when the 3HV content is increased. In particular, the flexibility is not improved to such an extent required for its use in films and the like. Thus, it has been used only in the field of rigid shaped articles such as shampoo bottles and disposable razor grips.
Under such circumstances, for making up the above-mentioned drawbacks of the copolymer derived from 3HB and 3HV, copolymers containing, as a component, a hydroxyalkanoic acid other than 3HB and 3HV such as 3-hydroxypropionic acid (hereinafter referred to briefly as “3HP”), 3-hydroxyhexanoic acid (hereinafter referred to briefly as “3HH”), 3-hydroxyoctanoic acid (hereinafter referred to briefly as “3HO”), 3-hydroxynonanoic acid (hereinafter referred to briefly as “3HN”), 3-hydroxydecanoic acid (hereinafter referred to briefly as “3HD”) or 3-hydroxydodecanoic acid (hereinafter referred to briefly as “3HDD”) are intensively studied (Poirier, Y., Nawrath C., Somerville C, BIO/TECHNOLOGY, 13, 142–150, 1995).
Among them, noteworthy studies are those on a copolyester comprising 3HB and 3HH units, particularly a copolymer P(3HB-co-3HH) derived only from 3HB and 3HH, and on a production method thereof (Japanese Kokai Publication Hei-05-93049 and Japanese Kokai Publication Hei-07-265065). The production methods of copolyesters such as P(3HB-co-3HH) described in these patent documents comprise a fermentation production from fatty acids such as oleic acid or oils and fats such as olive oil by using Aeromonas caviae isolated from soil.
A study regarding characteristics of P (3HB-co-3HH) has also been conducted (Y. Doi, S. Kitamura, H. Abe, Macromolecules 28, 4822–4823, 1995). This document reports a fermentation production of P (3HB-co-3HH) with a 3HH content of 11 to 19 mol % by culturing A. caviae with a fatty acid of not less than 12 carbon atoms. The result shows that, as the 3HH content increases, P(3HB-co-3HH) exhibits a gradual increase of flexibility from the hard and brittle characteristics of P (3HB) and finally shows more flexibility than P(3HB-co-3HV).
Additionally, it was reported that a polyhydroxyalkanoic acid(PHA) synthase gene from A. caviae was cloned and introduced into R. eutropha having an accumulating ability of polyhydroxybutyric acid(PHB) of not less than 90% to generate a recombinant strain, which was then used to produce P (3HB-co-3HH) using fatty acids as a carbon source (T. Fukui, Y. Doi, J. Bacteriol., vol. 179, No. 15, 4821–4830, 1997 and Japanese Kokai Publication Hei-10-108682). In these documents, it is reported that P (3HB-co-3HH) having the 3HH content of 10 to 20 mol % may be produced by using sodium octanoate as a carbon source.
Furthermore, a method has been recently disclosed which comprises using multiple carbon sources in producing a polyester using the above recombinant strain, and it was revealed that a carbon number of an oil or fat or a fatty acid used as a carbon source had an influence on the 3HH-content of P(3HB-co-3HH) (Japanese Kokai Publication 2001-340078).
If the 3HH content of P(3HB-co-3HH) can be controlled optionally in a wide range in the future, both hard copolymers and soft copolymers may be produced by fermentation, and such copolymers will find a broad range of applications, from chassis for TV-set, which is required to be hard, to a thread or a film, which are required to be flexible.
However, when a practical application of P(3HB-co-3HH) is considered, what becomes a barrier is the production cost. For example, in any methods which have already been disclosed, the productivity of P(3HB-co-3HH) is low and it is as much as 30 g/L. Additionally, a fatty acid having the carbon number of not less than 12, which is expensive as a carbon source, is used as the only carbon source, or an addition of an expensive fatty acid (hexanoic acid) is required to improve the 3HH content. Thus, it is scarcely possible to apply such technologies to an industrial production method of said polymer.
As described above, characteristics of P(3HB-co-3HH) are remarkably affected by the 3HH content. As a result of the investigation conducted by the present inventors, it is preferable to secure not less than 4 mol % of the 3HH content in order to enable wide applications of P(3HB-co-3HH). However, in the conventional culture methods, not only an expensive carbon source is required but also the productivity tends to be more deteriorated when trying to improve the 3HH content (see Japanese Kokai Publication 2001-340078).
Whereupon, it has been long awaited to develop a technology for realizing a high productivity of cells and polymer content in low cost, and from an industrial point of view, a technology capable of producing P(3HB-co-3HH) having the higher productivity, and preferably, a technology capable of producing P(3HB-co-3HH) having not less than 4 mol % of the 3HH content while securing that productivity.